Insight into the secondary structure of non-native proteins bound to a molecular chaperone alpha-crystallin - An isotope-edited infrared spectroscopic study

alpha-Crystallin, the major lens protein, acts as a molecular chaperone by preventing the aggregation of proteins damaged by heat and other stress con ditions. To characterize the backbone conformation of protein folding inter mediates that are recognized by the chaperone, we prepared the uniformly C- 13-labeled alpha A-crystallin. The labeling greatly reduced the overlapping between the conformation-sensitive amide I bands of alpha-crystallin and u nlabeled substrate proteins. This procedure has allowed us to gain insight into the secondary structure of alpha-crystallin-bound species, an understa nding which has previously been unattainable. Analysis of the infrared spec tra of two substrate proteins (gamma- and beta(L)-crystallins) indicates th at heat-destabilized conformers captured by alpha-crystallin are characteri zed by a high proportion of native-like secondary structure, In contrast to the chaperone-bound species, the same proteins subjected to heat treatment in the absence of alpha-crystallin preserve very little native secondary s tructure. These data show that cu-crystallin specifically recognizes very e arly intermediates on the denaturation pathway of proteins. These aggregati on-prone species are characterized by native-like secondary structure but c ompromised tertiary interactions. The experimental approach described in th is study can be further applied to probe the backbone conformation of prote ins bound to chaperones other than a-crystallin.